WO2007049487A1 - Substrate for analysis for use in raman spectroscopic analysis and substrate assembly for analysis - Google Patents

Substrate for analysis for use in raman spectroscopic analysis and substrate assembly for analysis Download PDF

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Publication number
WO2007049487A1
WO2007049487A1 PCT/JP2006/320703 JP2006320703W WO2007049487A1 WO 2007049487 A1 WO2007049487 A1 WO 2007049487A1 JP 2006320703 W JP2006320703 W JP 2006320703W WO 2007049487 A1 WO2007049487 A1 WO 2007049487A1
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Prior art keywords
substrate
analysis
analytical
raman
measurement
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PCT/JP2006/320703
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French (fr)
Japanese (ja)
Inventor
Toshihiro Ona
Shuichi Murakami
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Kyushu University, National University Corporation
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Application filed by Kyushu University, National University Corporation filed Critical Kyushu University, National University Corporation
Priority to EP06821905.4A priority Critical patent/EP1950556A4/en
Priority to CN2006800491295A priority patent/CN101351700B/en
Priority to JP2007542322A priority patent/JP4491616B2/en
Priority to US12/084,062 priority patent/US20090097021A1/en
Publication of WO2007049487A1 publication Critical patent/WO2007049487A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

Definitions

  • the present invention relates to an analysis substrate and an analysis substrate combination used for Raman spectroscopic analysis, and more particularly to an analysis substrate and an analysis substrate combination capable of analyzing a substance in a sample with high sensitivity. .
  • Raman spectroscopy is a spectroscopy that utilizes inelastic scattering (Raman scattering) of light. Since the Raman scattering is caused by each vibration of the molecule irradiated with light, a spectrum caused by the vibration inherent to the substance can be obtained by measuring the Raman scattering. Raman spectroscopy can analyze the state of samples in the same way as infrared spectroscopy, can measure samples of various shapes, is not particularly affected by moisture, has good wavenumber accuracy, and has a short measurement time. It has attracted attention from many fields such as agriculture. It is also considered to be used as a quality control method in the manufacturing industry. However, the detection sensitivity of Raman spectra is essentially not suitable for analysis of trace components that are low. In addition, there are no examples of application to clinical medicine for cancer diagnosis due to problems such as reproducibility and quantitativeness.
  • SERS surface-enhanced Raman spectroscopy
  • Raman spectroscopy is a spectroscopy that uses the phenomenon that Raman scattering of molecules adsorbed on metal nanostructures is enhanced more than usual.
  • the unevenness of the metal surface causes local enhancement of the electromagnetic field (electromagnetic field effect) and charge transfer to the vacant orbit of the adsorbed molecule in the locally enhanced field (chemical effect). It is thought that enhancement of Raman scattering occurs due to the superposition of two effects. Because SER S allows rapid analysis of trace components, research is being conducted for the purpose of detecting environmental hormones, pesticide residues and toxic components.
  • SERS surface-enhanced Raman spectroscopy
  • the inventors applied normal SERS, and when nano-order gold particles were formed on a glass substrate by vapor deposition, they were partially arranged narrowly at nano-order intervals, and the other portions were microscopic.
  • MERS transmission multiple enhanced Raman spectroscopy
  • the feature of this method is that multiple SERS measurements can be performed simultaneously by transmitting a single laser beam through multiple parallel substrates. In addition to this, it is conceivable to arrange multiple substrates in a certain volume of solution, and to enhance the light emission phenomenon and resonance with Raman scattered light.
  • the detection limit is 128 ppb with normal SERS, but 0.96 ppb with 5 enhancements (3 substrates).
  • the present invention is an analytical substrate used for Raman spectroscopic analysis, including a predetermined transparent substrate, metal particles adhering unevenly to the surface of the transparent substrate, and force. It is characterized by becoming.
  • the metal particles are densely packed at a nano-order interval in a predetermined region on the transparent substrate, and are dispersed at a micro-order interval in other regions.
  • the metal particles are made of gold, silver, copper, platinum, palladium, aluminum, titanium, or cobalt.
  • the transparent substrate is plate-shaped. [0010] Further, the transparent substrate is cylindrical.
  • the transparent substrate has a rectangular parallelepiped shape.
  • the present invention is characterized by comprising the above-mentioned cylindrical analysis substrate and at least one plate-shaped analysis substrate inserted into the cylindrical analysis substrate.
  • the present invention is characterized in that at least two cylindrical analysis substrates having different diameters are provided, and the analysis substrate having a small diameter is arranged inside the analysis substrate having a large diameter.
  • the present invention is characterized by comprising the above-mentioned rectangular parallelepiped analysis substrate and at least one plate-shaped analysis substrate inserted into the rectangular parallelepiped analysis substrate.
  • SERS measurement can be performed a plurality of times by transmission of laser light, and an enhancement greater than SERS measurement can be obtained.
  • FIG. 1 is a side view of an analytical substrate according to an embodiment of the present invention.
  • FIG. 2 is an atomic force micrograph of gold particles adhering to the analysis substrate disclosed in FIG.
  • FIG. 3 is a cross-sectional view showing a cylindrical substrate that has been used in conventional force Raman spectroscopy.
  • FIG. 4 is a cross-sectional view showing a substrate assembly for analysis according to another embodiment of the present invention.
  • FIG. 5 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
  • FIG. 6 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
  • FIG. 7 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
  • FIG. 8 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
  • FIG. 9 Concept explaining the principle of Raman spectroscopy using the analytical substrate combination disclosed in Fig. 6 FIG.
  • FIG. 10 is a diagram showing an application example of the analytical substrate assembly according to the present invention.
  • FIG. 11 is a diagram showing another application example of the analytical substrate assembly according to the present invention.
  • FIG. 12 is a view showing still another application example of the analytical substrate assembly according to the present invention.
  • FIG. 13 is a view showing still another application example of the analytical substrate assembly according to the present invention.
  • FIG. 14 is a view showing still another application example of the analytical substrate assembly according to the present invention.
  • FIG. 15 is a graph showing the relationship between Raman shift and Raman intensity obtained using the present invention.
  • FIG. 16 is a graph comparing the detection limit concentration and the detection sensitivity enhancement intensity when the number of enhancements is 0 and the number of enhancements using the present invention is 1.
  • FIG. 17 is a diagram comparing the detection limit concentration and the detection sensitivity increase intensity when the number of enhancements is 3 and the number of enhancements is 5 using the present invention.
  • FIG. 18 is a graph showing the relationship between the Raman peak intensity obtained using the present invention and the pyridine concentration.
  • FIG. 19 is a graph showing the relationship between Raman shift and Raman intensity obtained using the present invention for heavy metals.
  • FIG. 20 is a diagram comparing the detection limit concentration and the detection sensitivity enhancement strength when the number of enhancements is 0 and the number of enhancements using the present invention is 1 and 3 in the measurement of heavy metals.
  • FIG. 21 is a graph showing the relationship between Raman shift and Raman intensity obtained by using the present invention for bacteria.
  • FIG. 1 is a side view showing an example of an analysis substrate 1 in which gold particles 5 are attached to the surface of a glass substrate 3 having a plate-like glass force, which is a transparent substrate, and FIG. 2 shows this analysis substrate.
  • 1 is an atomic force micrograph of a partial surface of 1.
  • the atomic force micrograph in Fig. 2 is 4 X 4 / z square.
  • the white part in Fig. 2 is a region 7 where gold particles are dense, and the black part is a region 9 where gold particles are dispersed. In the dense region 7, the gold particles are close to each other in the nano order, and in the dispersion region 9, the gold particles are dispersed in the micro order.
  • the reason for this non-uniform distribution is that the laser beam used for Raman spectroscopy can pass through the analysis substrate 1 and further enhances Raman scattering in the dense region 7.
  • the metal particles used are not limited to gold, and may be metal particles such as silver, copper, platinum, palladium, aluminum, titanium, or corolet.
  • the metal particles are nanometer order metal nanoparticles including metal nanostructures such as nanorods.
  • n in the formula is the number of times light passes through the gold deposition surface. This can be paraphrased as the number of enhancements.
  • Ir (x) in the equation is the Raman scattering intensity when the Raman scattering of the sample generated on the X-th transmitted glass substrate reaches the detection part.
  • N (n) is the total amount of noise detected when transmitted n times, and D in the equation is the lower limit of Raman signal detection of the measuring device (detector).
  • the analytical substrate 1 is produced by a gold deposition method using a commercially available ion coater.
  • the glass substrate 3 was tilted and placed at a height of 7 cm at a target center force of 4.5 cm, and the deposition time was 30 minutes.
  • the deposition voltage at that time was 1.2 kV, and the deposition current was 5.5 mA.
  • the conditions must be set optimally for each ion coater used.
  • An analytical substrate made of cylindrical glass was also produced by the gold vapor deposition method as described above. Deposition conditions were as follows: Cylindrical glass was placed 1.5 cm away from the target and the deposition time was 30 minutes.
  • the analysis substrate may be produced using another apparatus connected with an ion coater.
  • a method of creating a target analysis substrate by creating a monomolecular film with holes on the order of nanometers at micrometer order intervals on a substrate, applying a metal nanocolloid, and then removing the monomolecular film. It is also possible to create a monomolecular film or metal colloid. It can also be created by a nanolithography method or a laser ablation method.
  • the above-mentioned analysis substrate 1 made of a plate-like glass cover has a size of about 3 X 15 mm square force 4 X 15 mm square in consideration of being placed inside the analysis substrate made of a cylindrical glass cover.
  • the cylindrical glass was used by cutting a commercially available NMR glass tube for measurement (5 mm ⁇ ) into a length of about 3 cm.
  • a glass container such as a rectangular parallelepiped glass cell is transparent. It may be used as a substrate. Of course, it is needless to say that the shape may be a cube. Furthermore, since it is only necessary to be transparent, these transparent substrates may be made of transparent plastic or sapphire glass.
  • FIG. 3 shows a substrate 11 used for normal Raman measurement (multiple enhancement times 0).
  • Sample T (about 300 ⁇ 1) is packed in a normal cylindrical glass 13.
  • Fig. 4 shows an analytical substrate assembly 21 used for SERS measurement (multiple enhancement times 1).
  • Sample ⁇ (approximately 300 ⁇ 1) is placed in a normal cylindrical gas.
  • a glass substrate 25b for analysis, which is filled in a glass 23 and further deposited with gold particles 25a, is disposed.
  • Fig. 5 shows an analytical substrate 31 used for MERS measurement (multiple enhancements 3 times).
  • Sample T (approximately 300 1) is packed in an analytical substrate 31 of cylindrical glass 35 on which gold particles 33 are deposited. It is.
  • FIG. 6 shows an analytical substrate assembly 41 used for MERS measurement (multiple enhancements 5 times).
  • Sample T (approximately 300 1) is contained in an analytical substrate 43 consisting of cylindrical glass 43b on which gold particles 43a are deposited.
  • an analytical substrate 45 of a plate-like glass 45b in which gold particles 45a are vapor-deposited is inserted.
  • Fig. 7 shows an analytical substrate assembly 51 used for MERS measurement (multiple enhancement times 7), and an analytical substrate consisting of cylindrical glass 53b with gold particles 53a deposited on sample T (approximately 3001).
  • An analysis substrate 55 made of a small-diameter cylindrical glass 55b with a gold particle 55a deposited therein is inserted into the inner 53.
  • Fig. 8 shows an analytical substrate assembly 61 used for MERS measurement (multiple enhancement times 9).
  • the analytical substrate 63 of plate-like glass 63b on which gold particles 63a are deposited in the configuration shown in Fig. 7 is calorie. is there.
  • FIG. 9 is a conceptual diagram for explaining the principle of enhancement when Raman spectroscopic analysis is performed using the analytical substrate assembly 41 shown in FIG.
  • the force combining the cylindrical analysis substrate 43 is described as a flat analysis substrate in FIG. 9 for convenience of explanation.
  • the left force laser beam L of the analysis substrate combination 41 is irradiated.
  • a part of the laser light L causes Raman scattering R by the measurement molecule S of the first (left) analysis substrate 43, and this is reflected to the light source (not shown).
  • the remaining part of the laser light L passes through the first analysis substrate 43 and is irradiated onto the second analysis substrate 45.
  • part of the laser beam L causes Raman scattering R by the measurement molecules S of the analysis substrate 45 and is reflected to the light source side.
  • the laser light L that has passed through the second analysis substrate 45 reaches the third (right side) analysis substrate 43, and here, Raman scattering R is generated by the measurement molecules S.
  • the light reflected from the second analysis substrate 45 to the light source side also causes Raman scattering R in the first analysis substrate 43, and the light reflected from the third analysis substrate 43 is the second.
  • Raman scattering R is also generated in the first analysis substrates 4 5 and 43.
  • the laser beam from the light source is reflected or reflected through the analysis substrates 43 and 45 at most five times. For this reason, detection sensitivity can be enhanced.
  • the analysis substrate assembly 71 shows an application example of the analysis substrate assembly.
  • a plurality of analysis substrates 73 made of a sheet glass 73a are arranged in parallel to each other and are accommodated in a predetermined container 75.
  • the surface of the analysis substrate 73 used here is provided with a coating layer 73c having a thickness of submicron order so as to cover the gold particles 73b.
  • the container 75 is filled with an aqueous solution containing the measurement molecule. Then, this analysis substrate combination 71 is irradiated with laser light, and the Raman scattering R is measured by a spectroscope.
  • FIG. 11 shows another application example of the analysis substrate assembly.
  • the analysis substrate combination 81 is an analysis substrate combination 81 using a meandering glass tube composed of a straight portion 83 and a curved portion 85, and the straight portions 53 are parallel to each other at a predetermined interval and on the same plane. Arranged above. Gold particles are deposited inside the glass tube.
  • the glass tube is filled with an aqueous solution containing a measurement molecule, the laser beam L is irradiated from a direction perpendicular to the straight portion 83, and the Raman scattering R is measured with a spectroscope.
  • FIG. 12 shows still another application example of the analysis substrate combination.
  • the analysis substrate combination 1101 is an analysis substrate combination 101 including a hollow rectangular parallelepiped or cube-shaped analysis substrate 103 and a plate-shaped analysis substrate 105 disposed inside the analysis substrate combination.
  • the analysis substrate 103 includes a transparent substrate 103b which is hollow and has a rectangular parallelepiped glass force, and gold particles 103a attached to the inner wall of the transparent substrate 103.
  • the plate-like analysis substrate 105 disposed inside is composed of a plate-like glass substrate 105b and gold particles 105a attached to the surface thereof.
  • the inside of the analysis substrate assembly 101 is filled with the sample T. In the analysis substrate assembly 101 having such a configuration, the gold particles pass up to five times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly described in FIG. 6 can be obtained.
  • FIG. 13 shows still another application example of the analysis substrate combination.
  • the analysis substrate combination 111 is an analysis substrate combination 111 composed of a hollow rectangular parallelepiped or cube-shaped analysis substrate 113 and a plate-shaped analysis substrate 115 disposed therein.
  • the analysis substrate 113 comprises a hollow, rectangular parallelepiped transparent glass substrate 113b having a glass power, and gold particles 113a attached to the inner wall of the transparent substrate 113b.
  • the plate-like analysis substrate 115 disposed inside comprises a plate-like glass substrate 105c and gold particles 115a and 115b attached to both side surfaces thereof.
  • the inside of the analysis substrate assembly 111 is filled with the sample T. Analysis of the configuration In the substrate assembly 111, gold particles are passed up to seven times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly shown in FIG. 7 can be obtained.
  • FIG. 14 shows still another application example of the analysis substrate assembly.
  • the analysis substrate combination 121 is an analysis substrate combination 121 composed of a cylindrical analysis substrate 123 and two plate-shaped analysis substrates 125 and 127 disposed therein.
  • the analysis substrate 123 includes a transparent substrate 123b made of a hollow and cylindrical glass glass, and a gold particle 123a attached to the inner wall of the transparent substrate 123b.
  • the plate-like analysis substrates 125 and 127 disposed inside serve as plate-like glass substrates 125b and 127b and gold particles 125a and 127a attached to the surfaces thereof, respectively.
  • the inside of the analysis substrate assembly 121 is filled with the sample T. In the analysis substrate assembly 121 having such a configuration, gold particles are passed through up to seven times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly described in FIG. 7 can be obtained. .
  • a commercially available Raman spectrometer was used for the Raman measurement. Measurement range of the Raman spectrometer is 300 ⁇ 2400cm 1. The excitation wavelength of the laser beam that excites the substrate for analysis was 785 nm, and the detector was measured using a CCD V and the number of integrations was 1 second X 5 (total 5 seconds). As a measurement method, a method using a commercially available probe was adopted. A pyridine aqueous solution was used as a sample. Pyridine aqueous solution is an enhanced confirmation component widely used in surface enhanced Raman spectroscopy (SERS) research. Ultrapure water was used for the preparation of the pyridine aqueous solution.
  • SERS surface enhanced Raman spectroscopy
  • a commercially available slab optical waveguide is used for thin film measurement.
  • the measurement is performed at an incident angle of 40 ° and the number of integrations of 1 second X 10 (10 seconds in total).
  • the optical waveguide was deposited on the opposite end face of the signal detector so that the laser beam was reflected, and then the upper face was deposited by the above gold deposition method.
  • FIG. 15 as an example, normal (enhanced zero impressions) for aqueous pyridine solution 1M Ramansu vector (A), 10- 4 normal Raman spectrum M aqueous pyridine (B- 1) and multiple enhancement Raman spectrum (enhancement The enhanced spectrum (B-2) by 3 times is shown.
  • pure water is filled into a cylindrical glass to obtain a Raman spectrum, and the background spectrum is corrected by subtracting the Raman spectrum force of the sample.
  • a peak is observed in the normal Raman spectrum (A) of a 1 M aqueous pyridine solution.
  • 10_ In the normal Raman spectrum (B-1) of 4 M pyridine aqueous solution, since the concentration is low, no peak is observed.
  • the Raman shift 1013cm 1 peak intensity attributed to the CH in-plane inflection vibration of pyridine on the X-axis in the Raman measurement of each enhancement or multiple enhancement frequency was obtained by plotting on the Y axis and creating a calibration curve. (See Figure 18).
  • the Raman peak intensity at a Raman shift of 1013 cm 1 was calculated by the following formula.
  • Peak intensity [Raman shift 1013 cm 1 scattering intensity (C—H in-plane bending vibration of pyridine)] [Raman shift 1013 cm 1 average of scattering intensity at both ends of peak]
  • the noise value shows no peaks in the Raman spectrum! ⁇
  • the root mean square (RMS) at 2300-2350 cm 1 was used.
  • the obtained detection limit signal value was compared with the calibration curve in Fig.
  • the detection limit enhancement was calculated by setting the detection limit concentration to 1 when the number of enhancements was zero. The results are shown in FIG. 16 and FIG. As a result, the detection limit for normal Raman measurement (0 enhancements) was 220 ppm, whereas for SERS (1 enhancement), the detection limit was 129 ppb, increasing the detection sensitivity by 1,700 (Fig. 16). reference). On the other hand, MERS (multiple enhancement times 5 times) was 0.96 ppb, and the detection sensitivity enhancement was 209,900 times that of normal Raman measurement (see Fig. 17). In other words, by using MERS, the enhancement of Raman scattering exceeding 10,000 times the detection sensitivity of a normal Raman spectrometer was confirmed.
  • the detection sensitivity enhancement increased as the number of multiple enhancements increased.
  • the light emission phenomenon caused by placing multiple substrates in a fixed volume of solution is resonant with Raman scattered light. It is also possible to reinforce. Opportunities for measurement molecules to be adsorbed to the substrate Therefore, the increase in detection sensitivity was suggested to increase more than simply proportional to the number of substrates. From the above, the effectiveness of multiple enhanced Raman spectroscopy was confirmed.
  • Heavy metals were also measured using the present invention.
  • -potassium chromate (K Cr 0) having hexavalent chromium was used as the heavy metal to be measured.
  • the measurement substrate was prepared by depositing gold on the substrate using a commercially available metal ion coater.
  • a commercially available process Raman spectrometer PI-200 manufactured by Process Instruments was used for the Raman measurement. Measurement range 300-2400cm Excitation wavelength 785, detector CCD, number of integrations 1 second X 5 (total 5 seconds), as a measurement method, for example, a commercially available probe (manufactured by Inphotonics) ) was used.
  • FIG 19 of 0.4 M - normal Raman spectrum of chromic acid aqueous potassium (A), 10- 4 M- chromic acid aqueous solution of potassium multiple enhanced number 3 times by enhancing the spectrum of (B-1: solid line), super multi enhancement number 3 times by enhancing the spectrum of pure water (B-2: dotted line) and 10- 3 M- chromic acid force
  • B-2 in Fig. 19 showed an increase in Raman scattering. Therefore, the relationship between the scattering intensity and concentration of 803 cm 1 in which Raman scattering enhancement was confirmed was plotted, a calibration curve was created, and the detection limit and enhancement intensity at each number of enhancements were calculated.
  • the noise value is at 2300-2350cm- 1 where no peaks are observed in the enhanced spectrum.
  • the root mean square (RMS) was used.
  • the detection limit at 0 enhancements was set to 1, and the detection limit for each enhancement was calculated. The results are shown in FIG. From these, it was found that MERS measurement of heavy metals is possible.
  • bacteria were also measured using the present invention.
  • Escherichia coli 0157 H: 7 and Staphylococcus aureus were used. After each culture was shaken overnight at 37 ° C, 10 ml of culture broth was repeatedly centrifuged (4 ° C, 8000 g, 5 minutes) and washed with sterile water 5 times. Then, it was mixed with 1 ml of sterilized water, diluted to an appropriate concentration, and used for measurement.
  • the measurement substrate was prepared by depositing silver on the substrate using a commercially available metal ion coater. For example, a commercially available process Raman spectrometer PI-200 (manufactured by Process Instruments) was used for the Raman measurement.
  • FIG. 21 shows Raman spectra of Escherichia coli (A) and Staphylococcus aureus (B) using MERS.
  • A in the figure, peaks were observed at 603 and 743 cm- 1
  • B in the figure, peaks were observed at 567 and 919 cm- 1 , so MERS measurement of food poisoning bacteria was performed. It was found that E. coli and Staphylococcus aureus could be distinguished.
  • amino acids, proteins, nucleic acids, toxins, fungi, polyamines, wood flour, paper, polyimide, graphite polycyclic aromatics, gas, plastics, conductive polymers, etc. can be measured using MERS. It is done.

Abstract

This invention provides a substrate or substrate assembly for Raman spectroscopic analysis that can analyze even a low-concentration substance with high sensitivity. The substrate (1) for use in Raman spectroscopic analysis comprises a predetermined transparent substrate (3) and metal particles (5) unevenly deposited on a surface of the transparent substrate (3).

Description

明 細 書  Specification
ラマン分光分析に用いる分析用基板及び分析用基板組合体  Analytical substrate and analytical substrate assembly used for Raman spectroscopy
技術分野  Technical field
[0001] 本発明は、ラマン分光分析に用いる分析用基板及び分析用基板組合体に係り、特 に、試料内の物質を高感度に分析することができる分析用基板及び分析用基板組 合体に関する。  TECHNICAL FIELD [0001] The present invention relates to an analysis substrate and an analysis substrate combination used for Raman spectroscopic analysis, and more particularly to an analysis substrate and an analysis substrate combination capable of analyzing a substance in a sample with high sensitivity. .
背景技術  Background art
[0002] ラマン分光法は、光の非弾性散乱 (ラマン散乱)を利用した分光法である。ラマン散 乱は、光を照射した分子の各振動に起因するため、このラマン散乱を測定することに より、物質固有の振動に起因するスペクトルを得ることができる。ラマン分光法は赤外 分光と同様に状態分析ができること、種々の形状の試料測定が可能で、特に水分の 影響を受けないこと、波数精度が良いこと、測定時間が短いことなど力 工学、薬学 および農学分野など多くの分野から注目されている。また、製造業における品質管理 手法としての利用も考えられている。しかし、ラマンスペクトルの検出感度は本質的に は低ぐ微量成分の分析には適していない。また、再現性や定量性などの問題から 癌診断の臨床医学に応用された例もない。  [0002] Raman spectroscopy is a spectroscopy that utilizes inelastic scattering (Raman scattering) of light. Since the Raman scattering is caused by each vibration of the molecule irradiated with light, a spectrum caused by the vibration inherent to the substance can be obtained by measuring the Raman scattering. Raman spectroscopy can analyze the state of samples in the same way as infrared spectroscopy, can measure samples of various shapes, is not particularly affected by moisture, has good wavenumber accuracy, and has a short measurement time. It has attracted attention from many fields such as agriculture. It is also considered to be used as a quality control method in the manufacturing industry. However, the detection sensitivity of Raman spectra is essentially not suitable for analysis of trace components that are low. In addition, there are no examples of application to clinical medicine for cancer diagnosis due to problems such as reproducibility and quantitativeness.
[0003] ラマン分光法を改良した SERS (表面増強ラマン分光法）は金属ナノ構造体に吸着 した分子のラマン散乱が通常よりも増強する現象を用いた分光法である。原理として 、分子が金属表面に吸着すると、金属表面の凹凸により電磁場の局所的増強 (電磁 場効果)と、局所的に増強された場における吸着分子の空軌道への電荷移動 (化学 効果)の 2つの効果の重畳により、ラマン散乱の増強が起きると考えられている。 SER Sにより迅速に微量成分の分析が可能なことから、環境ホルモン、残留農薬および有 毒成分などの検出の目的のために研究が行われている。  [0003] SERS (surface-enhanced Raman spectroscopy), which is an improvement of Raman spectroscopy, is a spectroscopy that uses the phenomenon that Raman scattering of molecules adsorbed on metal nanostructures is enhanced more than usual. In principle, when a molecule is adsorbed on a metal surface, the unevenness of the metal surface causes local enhancement of the electromagnetic field (electromagnetic field effect) and charge transfer to the vacant orbit of the adsorbed molecule in the locally enhanced field (chemical effect). It is thought that enhancement of Raman scattering occurs due to the superposition of two effects. Because SER S allows rapid analysis of trace components, research is being conducted for the purpose of detecting environmental hormones, pesticide residues and toxic components.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] 上記したように、微量成分分析法の一つである表面増強ラマン分光法 (SERS)は、 金属ナノ構造体に吸着した化学分子によるラマン散乱の増強程度が最大 10,000倍 になる現象を用いた分光法である。従来では、金属粒子を付着させた基板を試料中 に入れ、これに単一のレーザ光を照射し、反射した散乱光を検出していた。このため 、一回の測定で一度し力 SERSは発生せず、理論的な検出限界に拘束されていた。 また、基板に付着させる金属の種類の違いにより、増強を起こすィ匕学物質が異なるこ とが報告されており、水溶液中の多成分の同時検出が困難であるという問題点があ つた o [0004] As described above, surface-enhanced Raman spectroscopy (SERS), one of the trace component analysis methods, can increase Raman scattering by chemical molecules adsorbed on metal nanostructures up to 10,000 times. It is a spectroscopic method using the phenomenon. Conventionally, a substrate on which metal particles are attached is placed in a sample, and a single laser beam is irradiated on the substrate to detect reflected scattered light. For this reason, the force SERS was not generated once in one measurement, and was bound to the theoretical detection limit. In addition, it has been reported that chemical substances that cause enhancement differ depending on the type of metal attached to the substrate, which makes it difficult to simultaneously detect multiple components in an aqueous solution.
課題を解決するための手段  Means for solving the problem
[0005] 発明者らは、通常の SERSを応用し、ナノオーダーの金粒子を蒸着によりガラス基 板上に形成する際に、部分的にナノオーダーの間隔で狭く配置し、他の部分はマイ クロオーダーの間隔で広く配置した複数の基板を平行に配置し、レーザ光を通過さ せながら、更に反射光を検出することにより、多重に近接場も用い、 SERS測定を行 う方法を開発した。これは、透過型多重増強ラマン分光法 (MERS)と呼ぶことにする 。本手法の特長は、単一のレーザ光を平行に配置した複数の基板に透過させること により、複数回の SERS測定が同時に可能となることである。これにカ卩えて、一定容 量の溶液中に複数の基板を配置することによる、発光現象やラマン散乱光との共鳴 による増強も考えられる。また、複数の基板を用いる場合には、測定分子が基板に吸 着する機会が増加するため、増強、感度は基板の枚数に単純に比例する以上に増 加する。ピリジンの測定例では、通常の SERSでは検出限界が 128ppbであるのに対 し増強回数 5回（基板 3枚)では 0.96ppbと 、う結果が得られて 、る。  [0005] The inventors applied normal SERS, and when nano-order gold particles were formed on a glass substrate by vapor deposition, they were partially arranged narrowly at nano-order intervals, and the other portions were microscopic. Developed a method to perform SERS measurement using multiple near-fields by detecting multiple reflected light while passing multiple lasers arranged in parallel at a cross-order interval in parallel. . This is called transmission multiple enhanced Raman spectroscopy (MERS). The feature of this method is that multiple SERS measurements can be performed simultaneously by transmitting a single laser beam through multiple parallel substrates. In addition to this, it is conceivable to arrange multiple substrates in a certain volume of solution, and to enhance the light emission phenomenon and resonance with Raman scattered light. In addition, when a plurality of substrates are used, the opportunity for the measurement molecules to be adsorbed on the substrate increases, so that the enhancement and sensitivity increase more than simply proportional to the number of substrates. In the measurement example of pyridine, the detection limit is 128 ppb with normal SERS, but 0.96 ppb with 5 enhancements (3 substrates).
[0006] 上記のような結果を得るために、本発明では、ラマン分光分析に用いる分析用基板 であって、所定の透明基板と、この透明基板の表面に不均一に付着した金属粒子と 力 なることを特徴とする。  [0006] In order to obtain the results as described above, the present invention is an analytical substrate used for Raman spectroscopic analysis, including a predetermined transparent substrate, metal particles adhering unevenly to the surface of the transparent substrate, and force. It is characterized by becoming.
[0007] また、前記金属粒子は、前記透明基板上の所定の領域ではナノオーダーの間隔で 密集し、それ以外の領域ではマイクロオーダーの間隔で分散して 、ることを特徴とす る。  [0007] Further, the metal particles are densely packed at a nano-order interval in a predetermined region on the transparent substrate, and are dispersed at a micro-order interval in other regions.
[0008] また、前記金属粒子は金、銀、銅、白金、パラジウム、アルミニウム、チタン又はコバ ルトからなることを特徴とする。  [0008] Further, the metal particles are made of gold, silver, copper, platinum, palladium, aluminum, titanium, or cobalt.
[0009] また、前記透明基板は、板状であることを特徴とする。 [0010] また、前記透明基板は、円筒状であることを特徴とする。 [0009] Further, the transparent substrate is plate-shaped. [0010] Further, the transparent substrate is cylindrical.
[0011] また、前記透明基板は、直方体状であることを特徴とする。 [0011] Further, the transparent substrate has a rectangular parallelepiped shape.
[0012] また、少なくとも 2枚の板状の上記分析用基板を相互に平行に配列したことを特徴 とする。  [0012] Further, it is characterized in that at least two plate-like analysis substrates are arranged in parallel to each other.
[0013] また、円筒状の上記分析用基板と、この円筒状の分析用基板の内部に挿入される 少なくとも 1枚の上記板状の分析用基板とからなることを特徴とする。  [0013] Further, the present invention is characterized by comprising the above-mentioned cylindrical analysis substrate and at least one plate-shaped analysis substrate inserted into the cylindrical analysis substrate.
[0014] また、直径の異なる少なくとも 2つの円筒状の上記分析用基板を備え、直径の大き な分析用基板の内部に直径の小さな分析用基板を配置したことを特徴とする。 [0014] Further, the present invention is characterized in that at least two cylindrical analysis substrates having different diameters are provided, and the analysis substrate having a small diameter is arranged inside the analysis substrate having a large diameter.
[0015] また、上記直方体状の分析用基板と、この直方体状の分析用基板の内部に挿入さ れる少なくとも 1枚の板状の上記分析用基板とからなることを特徴とする。 [0015] Further, the present invention is characterized by comprising the above-mentioned rectangular parallelepiped analysis substrate and at least one plate-shaped analysis substrate inserted into the rectangular parallelepiped analysis substrate.
[0016] 更に、前記複数の分析用基板には、相互に異なる金属粒子が付着されていること を特徴とする。 [0016] Further, different metal particles are attached to the plurality of analysis substrates.
発明の効果  The invention's effect
[0017] 本発明によれば、レーザ光の透過による複数回の SERS測定が可能で、 SERS測 定以上の増強度が得られる。また、基板数の増減により増強程度の制御が可能であ り、複数の異種金属膜基板を組み合わせることによる多成分検出が可能である点が 、既存技術に対して優位である。  [0017] According to the present invention, SERS measurement can be performed a plurality of times by transmission of laser light, and an enhancement greater than SERS measurement can be obtained. In addition, it is possible to control the degree of enhancement by increasing or decreasing the number of substrates, and it is advantageous over existing technologies in that multi-component detection is possible by combining multiple dissimilar metal film substrates.
図面の簡単な説明  Brief Description of Drawings
[0018] [図 1]本発明の一実施形態に係る分析用基板の側面図である。 FIG. 1 is a side view of an analytical substrate according to an embodiment of the present invention.
[図 2]図 1に開示した分析用基板に付着している金粒子の原子間力顕微鏡写真であ る。  FIG. 2 is an atomic force micrograph of gold particles adhering to the analysis substrate disclosed in FIG.
[図 3]従来力 ラマン分光法に用いられてきた円筒状基板を示す断面図である。  FIG. 3 is a cross-sectional view showing a cylindrical substrate that has been used in conventional force Raman spectroscopy.
[図 4]本発明の他の実施形態に係る分析用基板組合体を示す断面図である。  FIG. 4 is a cross-sectional view showing a substrate assembly for analysis according to another embodiment of the present invention.
[図 5]本発明の更に他の実施形態に係る分析用基板組合体を示す断面図である。  FIG. 5 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
[図 6]本発明の更に他の実施形態に係る分析用基板組合体を示す断面図である。  FIG. 6 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
[図 7]本発明の更に他の実施形態に係る分析用基板組合体を示す断面図である。  FIG. 7 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
[図 8]本発明の更に他の実施形態に係る分析用基板組合体を示す断面図である。  FIG. 8 is a cross-sectional view showing an analytical substrate assembly according to still another embodiment of the present invention.
[図 9]図 6に開示した分析用基板組合体を用いたラマン分光の原理を説明する概念 図である。 [Fig. 9] Concept explaining the principle of Raman spectroscopy using the analytical substrate combination disclosed in Fig. 6 FIG.
[図 10]本発明に係る分析用基板組合体の応用例を示す図である。  FIG. 10 is a diagram showing an application example of the analytical substrate assembly according to the present invention.
[図 11]本発明に係る分析用基板組合体の他の応用例を示す図である。  FIG. 11 is a diagram showing another application example of the analytical substrate assembly according to the present invention.
[図 12]本発明に係る分析用基板組合体の更に他の応用例を示す図である。  FIG. 12 is a view showing still another application example of the analytical substrate assembly according to the present invention.
[図 13]本発明に係る分析用基板組合体の更に他の応用例を示す図である。  FIG. 13 is a view showing still another application example of the analytical substrate assembly according to the present invention.
[図 14]本発明に係る分析用基板組合体の更に他の応用例を示す図である。  FIG. 14 is a view showing still another application example of the analytical substrate assembly according to the present invention.
[図 15]本発明を用いて得られたラマンシフトとラマン強度との関係を示すグラフである  FIG. 15 is a graph showing the relationship between Raman shift and Raman intensity obtained using the present invention.
[図 16]増強回数 0回と本発明を用いた増強回数 1回による検出限界濃度と検出感度 増強度を比較した図である。 FIG. 16 is a graph comparing the detection limit concentration and the detection sensitivity enhancement intensity when the number of enhancements is 0 and the number of enhancements using the present invention is 1.
[図 17]本発明を用いた増強回数 3回と増強回数 5回による検出限界濃度と検出感度 増強度を比較した図である。  FIG. 17 is a diagram comparing the detection limit concentration and the detection sensitivity increase intensity when the number of enhancements is 3 and the number of enhancements is 5 using the present invention.
[図 18]本発明を用いて得られたラマンピーク強度とピリジン濃度との関係を示すダラ フである。  FIG. 18 is a graph showing the relationship between the Raman peak intensity obtained using the present invention and the pyridine concentration.
[図 19]重金属に対して本発明を用いて得られたラマンシフトとラマン強度との関係を 示すグラフである。  FIG. 19 is a graph showing the relationship between Raman shift and Raman intensity obtained using the present invention for heavy metals.
[図 20]重金属の測定において、増強回数 0回と本発明を用いた増強回数 1回と 3回 による検出限界濃度と検出感度増強度を比較した図である。  FIG. 20 is a diagram comparing the detection limit concentration and the detection sensitivity enhancement strength when the number of enhancements is 0 and the number of enhancements using the present invention is 1 and 3 in the measurement of heavy metals.
[図 21]菌に対して本発明を用いて得られたラマンシフトとラマン強度との関係を示す グラフである。  FIG. 21 is a graph showing the relationship between Raman shift and Raman intensity obtained by using the present invention for bacteria.
符号の説明 Explanation of symbols
1 分析用基板 1 Analysis substrate
3 ガラス基板 3 Glass substrate
5 金粒子 5 gold particles
7 密集領域 7 Dense area
9 分散領域 9 Distributed area
21 分析用基板組合体 21 Analytical substrate assembly
23 円筒状ガラス基板 a 金粒子23 Cylindrical glass substrate a gold particles
b 板状ガラス基板 b Sheet glass substrate
分析用ガラス基板  Glass substrate for analysis
金粒子  Gold particles
円筒状ガラス基板  Cylindrical glass substrate
分析用基板組合体 円筒状分析用基板 板状分析用基板  Analysis substrate assembly Cylindrical analysis substrate Plate analysis substrate
分析用基板組合体 円筒状分析用基板 (大直径) 円筒状分析用基板 (小直径) 分析用基板組合体 板状分析用基板  Analysis substrate combination Cylindrical analysis substrate (large diameter) Cylindrical analysis substrate (small diameter) Analysis substrate combination Plate analysis substrate
分析用基板組合体 板状分析用基板  Analysis substrate assembly Plate analysis substrate
谷器  Trough
分析用基板組合体 Analysis board assembly
1 分析用基板組合体1 Analytical board assembly
3 分析用基板3 Analysis substrate
5 分析用基板5 Analysis substrate
1 分析用基板組合体1 Analytical board assembly
3 分析用基板3 Analysis substrate
5 分析用基板5 Analysis substrate
1 分析用基板組合体1 Analytical board assembly
3 分析用基板3 Analysis substrate
5 分析用基板5 Analysis substrate
7 分析用基板 7 Analysis substrate
レーザ光 R ラマン散乱 Laser light R Raman scattering
s 測定分子  s Measuring molecule
T 試料  T sample
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0020] 次に、図面を用いて本発明の一実施形態について説明する。  [0020] Next, an embodiment of the present invention will be described with reference to the drawings.
[0021] 図 1は、透明基板である板状ガラス力もなるガラス基板 3の表面に金粒子 5を付着さ せた分析用基板 1の例を示す側面図であり、図 2はこの分析用基板 1の一部表面の 原子間力顕微鏡写真である。図 1においては、説明の便宜上、蒸着された金粒子 5 の層の厚さを誇張して記載している。図 2の原子間力顕微鏡写真は、 4 X 4 /z m角の 大きさである。図 2中の白い部分は金粒子が密集している領域 7であり、黒い部分は 金粒子が分散して 、る領域 9である。密集領域 7では金粒子同士がナノオーダーで 近接しており、分散領域 9では金粒子同士はマイクロオーダーで分散している。この ような不均一な分布としたのは、ラマン分光に用いるレーザ光が分析用基板 1を透過 することができ、さらに密集領域 7においてラマン散乱の増強を起こさせるためである 。尚、用いる金属粒子は金に限定されるものではなぐ銀、銅、白金、パラジウム、ァ ルミ二ゥム、チタン又はコノ レトなどの金属粒子であってもよい。また、金属粒子は、 ナノロッド等の金属ナノ構造体も含むナノメートルオーダーの金属ナノ粒子である。 FIG. 1 is a side view showing an example of an analysis substrate 1 in which gold particles 5 are attached to the surface of a glass substrate 3 having a plate-like glass force, which is a transparent substrate, and FIG. 2 shows this analysis substrate. 1 is an atomic force micrograph of a partial surface of 1. In FIG. 1, for convenience of explanation, the thickness of the deposited gold particles 5 is exaggerated. The atomic force micrograph in Fig. 2 is 4 X 4 / z square. The white part in Fig. 2 is a region 7 where gold particles are dense, and the black part is a region 9 where gold particles are dispersed. In the dense region 7, the gold particles are close to each other in the nano order, and in the dispersion region 9, the gold particles are dispersed in the micro order. The reason for this non-uniform distribution is that the laser beam used for Raman spectroscopy can pass through the analysis substrate 1 and further enhances Raman scattering in the dense region 7. The metal particles used are not limited to gold, and may be metal particles such as silver, copper, platinum, palladium, aluminum, titanium, or corolet. The metal particles are nanometer order metal nanoparticles including metal nanostructures such as nanorods.
[0022] 金粒子の密集及び分散の度合いにつ!、ては種々の設定が可能である力 物質の 濃度が低濃度の試料であっても確実に分析できるようにするために、以下の条件を 満たすことが必要である。  [0022] Various settings are possible for the degree of density and dispersion of gold particles. In order to ensure that even a sample with a low concentration of substance can be analyzed, the following conditions are satisfied. It is necessary to satisfy
[0023] [数 1]  [0023] [Equation 1]
∑ n=l Ir(n) ∑ n = l Ir (n)
> D  > D
N(n)  N (n)
[0024] ここでは、試料中における測定分子の濃度が、通常のラマン分光分析では不可能 な場合を想定している。先ず、式中の nは光が金蒸着面を透過する回数である。これ は増強回数と言い換えることもできる。また、式中の Ir(x)は X番目に透過したガラス 基板で発生した試料のラマン散乱が検出部に到達した時のラマン散乱強度、式中の N (n)は n回透過したときに検出されるノイズの総量、そして式中の Dは測定装置 (検 出器)のラマンシグナル検出下限値である。 [0024] Here, it is assumed that the concentration of the molecule to be measured in the sample is not possible by ordinary Raman spectroscopic analysis. First, n in the formula is the number of times light passes through the gold deposition surface. This can be paraphrased as the number of enhancements. In addition, Ir (x) in the equation is the Raman scattering intensity when the Raman scattering of the sample generated on the X-th transmitted glass substrate reaches the detection part. N (n) is the total amount of noise detected when transmitted n times, and D in the equation is the lower limit of Raman signal detection of the measuring device (detector).
[0025] 本実施形態に係る分析用基板 1は、市販されているイオンコーターを用いて、金蒸 着法によって作製される。実際の蒸着工程では、ガラス基板 3をターゲット中心部力 4.5cm離れた高さ 7cmの位置に傾けて配置し、蒸着時間 30分で行った。そのときの 蒸着電圧は 1.2kVであり、蒸着電流は 5.5mAとした。但し、当該条件は用いるイオン コーターごとに最適に設定する必要がある。また、円筒状ガラスからなる分析用基板 も上記と同様に金蒸着法により作製した。蒸着条件は、円筒状ガラスをターゲット真 上から 1.5cm離れた位置に配置し、蒸着時間 30分で行った。そのときの蒸着電圧は 1 .2kVであり、蒸着電流は 5.5mAとした。尚、本発明では、不均一な金属（例えば、金 )粒子の分布が形成されればよいので、イオンコーターでなぐ他の装置を用いて分 析用基板を作製してもよ ヽ。例えばナノメートルオーダーの穴をマイクロメートルオー ダー間隔であけた状態の単分子膜を基板に作成し、金属ナノコロイドを塗布し、その 後単分子膜を除き、目的分析基板を作成する方法のように単分子膜や金属コロイド を用い作成することも可能である。また、ナノリソグラフィ一法やレーザーアブレーショ ン法による作成も可能である。  [0025] The analytical substrate 1 according to the present embodiment is produced by a gold deposition method using a commercially available ion coater. In the actual vapor deposition process, the glass substrate 3 was tilted and placed at a height of 7 cm at a target center force of 4.5 cm, and the deposition time was 30 minutes. The deposition voltage at that time was 1.2 kV, and the deposition current was 5.5 mA. However, the conditions must be set optimally for each ion coater used. An analytical substrate made of cylindrical glass was also produced by the gold vapor deposition method as described above. Deposition conditions were as follows: Cylindrical glass was placed 1.5 cm away from the target and the deposition time was 30 minutes. The deposition voltage at that time was 1.2 kV, and the deposition current was 5.5 mA. In the present invention, since an uneven distribution of metal (for example, gold) particles only needs to be formed, the analysis substrate may be produced using another apparatus connected with an ion coater. For example, a method of creating a target analysis substrate by creating a monomolecular film with holes on the order of nanometers at micrometer order intervals on a substrate, applying a metal nanocolloid, and then removing the monomolecular film. It is also possible to create a monomolecular film or metal colloid. It can also be created by a nanolithography method or a laser ablation method.
[0026] 上記した板状ガラスカゝらなる分析用基板 1は、円筒状ガラスカゝらなる分析用基板の 内部に配置することも考慮し、 3 X 15mm角力 4 X 15mm角程度の大きさのものを用 いた。また、円筒状ガラスは、市販の測定用 NMRガラスチューブ (5mm φ )を長さ約 3cmに切断して用いた。尚、上記説明では板状若しく円筒状の透明基板を用いた分 析用基板について記載しているが、本発明はこれに限定されるものではなぐ直方体 形状のガラスセルなどのガラス容器を透明基板として用いてもよい。もちろん、立方体 形状であっても良いことは言うまでもない。更に、透明であればよいので、これらの透 明基板を透明プラスチック或いはサファイアガラスなどで作製してもよ ヽ。  [0026] The above-mentioned analysis substrate 1 made of a plate-like glass cover has a size of about 3 X 15 mm square force 4 X 15 mm square in consideration of being placed inside the analysis substrate made of a cylindrical glass cover. Was used. The cylindrical glass was used by cutting a commercially available NMR glass tube for measurement (5 mmφ) into a length of about 3 cm. In the above description, the analysis substrate using a plate-like or cylindrical transparent substrate is described, but the present invention is not limited to this, and a glass container such as a rectangular parallelepiped glass cell is transparent. It may be used as a substrate. Of course, it is needless to say that the shape may be a cube. Furthermore, since it is only necessary to be transparent, these transparent substrates may be made of transparent plastic or sapphire glass.
[0027] 次に、分析用基板及び分析用基板組合体の様々な具体例について説明する。図 3は通常のラマン測定 (多重増強回数 0回）に用いる基板 11であり、試料 T (約 300 μ 1 )を通常の円筒状ガラス内 13に充填したものである。図 4は SERS測定 (多重増強回 数 1回）に用いる分析用基板組合体 21であり、試料 Τ (約 300 ^ 1)を通常の円筒状ガ ラス 23内に充填し、更に金粒子 25aが蒸着された板状ガラス 25bの分析用基板 25を 配置したものである。図 5は MERS測定 (多重増強回数 3回）に用いる分析用基板 31 であり、試料 T (約 300 1)を金粒子 33が蒸着された円筒状ガラス 35の分析用基板 内 31に充填したものである。図 6は MERS測定 (多重増強回数 5回）に用いる分析用 基板組合体 41であり、試料 T (約 300 1)を金粒子 43aが蒸着された円筒状ガラス 43 bからなる分析用基板 43内に充填し、更にその内部に金粒子 45aが蒸着された板状 ガラス 45bの分析用基板 45を挿入したものである。図 7は、 MERS測定 (多重増強回 数 7回）に用いる分析用基板組合体 51であり、試料 T (約 300 1)を金粒子 53aが蒸 着された円筒状ガラス 53bからなる分析用基板内 53に充填し、更にその内部に金粒 子 55aが蒸着された小直径の円筒状ガラス 55bからなる分析用基板 55を挿入したも のである。図 8は、 MERS測定 (多重増強回数 9回）に用いる分析用基板組合体 61 であり、図 7の構成に金粒子 63aが蒸着された板状ガラス 63bの分析用基板 63をカロ えたものである。 Next, various specific examples of the analysis substrate and the analysis substrate combination will be described. Fig. 3 shows a substrate 11 used for normal Raman measurement (multiple enhancement times 0). Sample T (about 300 µ 1) is packed in a normal cylindrical glass 13. Fig. 4 shows an analytical substrate assembly 21 used for SERS measurement (multiple enhancement times 1). Sample Τ (approximately 300 ^ 1) is placed in a normal cylindrical gas. A glass substrate 25b for analysis, which is filled in a glass 23 and further deposited with gold particles 25a, is disposed. Fig. 5 shows an analytical substrate 31 used for MERS measurement (multiple enhancements 3 times). Sample T (approximately 300 1) is packed in an analytical substrate 31 of cylindrical glass 35 on which gold particles 33 are deposited. It is. Figure 6 shows an analytical substrate assembly 41 used for MERS measurement (multiple enhancements 5 times). Sample T (approximately 300 1) is contained in an analytical substrate 43 consisting of cylindrical glass 43b on which gold particles 43a are deposited. And an analytical substrate 45 of a plate-like glass 45b in which gold particles 45a are vapor-deposited is inserted. Fig. 7 shows an analytical substrate assembly 51 used for MERS measurement (multiple enhancement times 7), and an analytical substrate consisting of cylindrical glass 53b with gold particles 53a deposited on sample T (approximately 3001). An analysis substrate 55 made of a small-diameter cylindrical glass 55b with a gold particle 55a deposited therein is inserted into the inner 53. Fig. 8 shows an analytical substrate assembly 61 used for MERS measurement (multiple enhancement times 9). The analytical substrate 63 of plate-like glass 63b on which gold particles 63a are deposited in the configuration shown in Fig. 7 is calorie. is there.
図 9は、図 6に示す分析用基板組合体 41を用いてラマン分光分析をした場合の増 強原理を説明する概念図である。図 6では円筒状の分析用基板 43を組み合わせて いる力 図 9では説明の便宜上全て平面状の分析用基板で説明する。この図に示す ように、分析用基板組合体 41の左方力 レーザ光 Lが照射される。レーザ光 Lの一部 は第 1 (左側）の分析用基板 43の測定分子 Sによってラマン散乱 Rを生じさせ、これを 図示しない光源側に反射させる。一方、レーザ光 Lの残りの部分は第 1の分析用基 板 43を通過して、第 2の分析用基板 45に照射される。ここでもレーザ光 Lの一部は 分析用基板 45の測定分子 Sによってラマン散乱 Rを生じさせ、光源側に反射される。 更に、第 2の分析用基板 45を通過したレーザ光 Lは、第 3 (右側）の分析用基板 43に 到達し、ここでも測定分子 Sによってラマン散乱 Rを生じさせる。これらに加え、第 2の 分析用基板 45から光源側に反射する光は第 1の分析用基板 43でもラマン散乱 Rを 生じさせ、更に、第 3の分析用基板 43から反射する光は第 2及び第 1の分析用基板 4 5, 43でもラマン散乱 Rを生じさせることとなる。このように、光源からのレーザ光しが 反射するまでには、分析用基板 43, 45を最高 5回通過若しくは反射することになる。 このようなことから、検出感度の増強が図られる。 [0029] 図 10は、分析用基板組合体の応用例を示す。当該分析用基板組合体 71では、板 状ガラス 73aからなる複数の分析用基板 73が相互に平行に配列され、これが所定の 容器 75に収容されているものである。ここで用いられる分析用基板 73の表面には、 金粒子 73bを覆うようにサブミクロンオーダーの厚さの被覆層 73cが施されている。容 器 75内には測定分子が含まれた水溶液が満たされている。そして、この分析用基板 組合体 71にレーザ光が照射され、ラマン散乱 Rを分光器によって測定する。 FIG. 9 is a conceptual diagram for explaining the principle of enhancement when Raman spectroscopic analysis is performed using the analytical substrate assembly 41 shown in FIG. In FIG. 6, the force combining the cylindrical analysis substrate 43 is described as a flat analysis substrate in FIG. 9 for convenience of explanation. As shown in this figure, the left force laser beam L of the analysis substrate combination 41 is irradiated. A part of the laser light L causes Raman scattering R by the measurement molecule S of the first (left) analysis substrate 43, and this is reflected to the light source (not shown). On the other hand, the remaining part of the laser light L passes through the first analysis substrate 43 and is irradiated onto the second analysis substrate 45. Here too, part of the laser beam L causes Raman scattering R by the measurement molecules S of the analysis substrate 45 and is reflected to the light source side. Further, the laser light L that has passed through the second analysis substrate 45 reaches the third (right side) analysis substrate 43, and here, Raman scattering R is generated by the measurement molecules S. In addition to these, the light reflected from the second analysis substrate 45 to the light source side also causes Raman scattering R in the first analysis substrate 43, and the light reflected from the third analysis substrate 43 is the second. In addition, Raman scattering R is also generated in the first analysis substrates 4 5 and 43. As described above, the laser beam from the light source is reflected or reflected through the analysis substrates 43 and 45 at most five times. For this reason, detection sensitivity can be enhanced. FIG. 10 shows an application example of the analysis substrate assembly. In the analysis substrate assembly 71, a plurality of analysis substrates 73 made of a sheet glass 73a are arranged in parallel to each other and are accommodated in a predetermined container 75. The surface of the analysis substrate 73 used here is provided with a coating layer 73c having a thickness of submicron order so as to cover the gold particles 73b. The container 75 is filled with an aqueous solution containing the measurement molecule. Then, this analysis substrate combination 71 is irradiated with laser light, and the Raman scattering R is measured by a spectroscope.
[0030] 図 11は、分析用基板組合体の他の応用例を示す。当該分析用基板組合体 81で は、直線部 83と曲線部 85からなる蛇行したガラス管を用いた分析用基板組合体 81 であり、直線部分 53が所定間隔を隔てて相互に平行で同一平面上に配列されてい る。ガラス管の内部には金粒子が蒸着されている。分析に際しては、ガラス管内に測 定分子を含む水溶液を満たし、直線部分 83に対して垂直な方向からレーザ光 Lが照 射され、ラマン散乱 Rを分光器で測定する。  FIG. 11 shows another application example of the analysis substrate assembly. The analysis substrate combination 81 is an analysis substrate combination 81 using a meandering glass tube composed of a straight portion 83 and a curved portion 85, and the straight portions 53 are parallel to each other at a predetermined interval and on the same plane. Arranged above. Gold particles are deposited inside the glass tube. In the analysis, the glass tube is filled with an aqueous solution containing a measurement molecule, the laser beam L is irradiated from a direction perpendicular to the straight portion 83, and the Raman scattering R is measured with a spectroscope.
[0031] 図 12は、分析用基板組合体の更に他の応用例を示す。当該分析用基板組合体 1 01は、中空の直方体若しくは立方体形状の分析用基板 103と、この内部に配置され る板状の分析用基板 105とからなる分析用基板組合体 101である。分析用基板 103 は中空で直方体状のガラス力もなる透明基板 103bと、この透明基板 103の内壁に 付着される金粒子 103aとからなる。また、内部に配置される板状の分析用基板 105 は、板状のガラス基板 105bとその表面に付着される金粒子 105aとからなる。分析用 基板組合体 101の内部には試料 Tが充填されて 、る。当該構成の分析用基板組合 体 101では、外部から光が入射して反射するまでに金粒子を最高 5回通過するので 、図 6に記載した分析用基板組合体と同様の効果が得られる。  FIG. 12 shows still another application example of the analysis substrate combination. The analysis substrate combination 1101 is an analysis substrate combination 101 including a hollow rectangular parallelepiped or cube-shaped analysis substrate 103 and a plate-shaped analysis substrate 105 disposed inside the analysis substrate combination. The analysis substrate 103 includes a transparent substrate 103b which is hollow and has a rectangular parallelepiped glass force, and gold particles 103a attached to the inner wall of the transparent substrate 103. The plate-like analysis substrate 105 disposed inside is composed of a plate-like glass substrate 105b and gold particles 105a attached to the surface thereof. The inside of the analysis substrate assembly 101 is filled with the sample T. In the analysis substrate assembly 101 having such a configuration, the gold particles pass up to five times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly described in FIG. 6 can be obtained.
[0032] 図 13は、分析用基板組合体の更に他の応用例を示す。当該分析用基板組合体 1 11は、中空の直方体若しくは立方体形状の分析用基板 113と、この内部に配置され る板状の分析用基板 115とからなる分析用基板組合体 111である。分析用基板 113 は中空で直方体状のガラス力 なる透明基板 113bと、この透明基板 113bの内壁に 付着される金粒子 113aとからなる。また、内部に配置される板状の分析用基板 115 は、板状のガラス基板 105cとその両側表面に付着される金粒子 115a、 115bとから なる。分析用基板組合体 111の内部には試料 Tが充填されている。当該構成の分析 用基板組合体 111では、外部から光が入射して反射するまでに金粒子を最高 7回通 過するので、図 7に記載した分析用基板組合体と同様の効果が得られる。 FIG. 13 shows still another application example of the analysis substrate combination. The analysis substrate combination 111 is an analysis substrate combination 111 composed of a hollow rectangular parallelepiped or cube-shaped analysis substrate 113 and a plate-shaped analysis substrate 115 disposed therein. The analysis substrate 113 comprises a hollow, rectangular parallelepiped transparent glass substrate 113b having a glass power, and gold particles 113a attached to the inner wall of the transparent substrate 113b. Further, the plate-like analysis substrate 115 disposed inside comprises a plate-like glass substrate 105c and gold particles 115a and 115b attached to both side surfaces thereof. The inside of the analysis substrate assembly 111 is filled with the sample T. Analysis of the configuration In the substrate assembly 111, gold particles are passed up to seven times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly shown in FIG. 7 can be obtained.
[0033] 図 14は、分析用基板組合体の更に他の応用例を示す。当該分析用基板組合体 1 21は、円筒状の分析用基板 123と、この内部に配置される 2枚の板状の分析用基板 125, 127とからなる分析用基板組合体 121である。分析用基板 123は中空で円筒 状のガラスカゝらなる透明基板 123bと、この透明基板 123bの内壁に付着される金粒 子 123aとからなる。また、内部に配置される板状の分析用基板 125、 127は、それぞ れ板状のガラス基板 125b、 127bとその表面に付着される金粒子 125a、 127aと力 なる。分析用基板組合体 121の内部には試料 Tが充填されている。当該構成の分析 用基板組合体 121では、外部から光が入射して反射するまでに金粒子を最高 7回通 過するので、図 7に記載した分析用基板組合体と同様の効果が得られる。  FIG. 14 shows still another application example of the analysis substrate assembly. The analysis substrate combination 121 is an analysis substrate combination 121 composed of a cylindrical analysis substrate 123 and two plate-shaped analysis substrates 125 and 127 disposed therein. The analysis substrate 123 includes a transparent substrate 123b made of a hollow and cylindrical glass glass, and a gold particle 123a attached to the inner wall of the transparent substrate 123b. In addition, the plate-like analysis substrates 125 and 127 disposed inside serve as plate-like glass substrates 125b and 127b and gold particles 125a and 127a attached to the surfaces thereof, respectively. The inside of the analysis substrate assembly 121 is filled with the sample T. In the analysis substrate assembly 121 having such a configuration, gold particles are passed through up to seven times before the light is incident and reflected from the outside, so that the same effect as the analysis substrate assembly described in FIG. 7 can be obtained. .
[0034] 次に、具体的な測定及び分析について説明する。ラマン測定には、市販されてい るラマン分光計を用いた。このラマン分光計の測定範囲は 300〜2400cm 1である。分 析用基板を励起するレーザ光の励起波長は 785nmであり、検出器は CCDを用 V、積 算回数 1秒 X 5 (合計 5秒）にて測定を行った。測定方法としては、市販されているプ ローブを用いる方法を採用した。また、試料としてはピリジン水溶液を用いた。ピリジ ン水溶液は表面増強ラマン分光法 (SERS)研究にぉ 、て広く用いられる増強確認 成分である。ピリジン水溶液の調製には超純水を用いた。薄膜測定には市販のスラ ブ型光導波路を用いる。尚、光導波路を用いたラマン測定では、上記の条件に加え 、入射角 40° 、積算回数 1秒 X 10 (合計 10秒）にて測定を行う。光導波路はシグナ ル検出部の反対側の端面をレーザ光が反射するように蒸着を施したのち、上面を上 記の金蒸着法で蒸着した。 Next, specific measurement and analysis will be described. A commercially available Raman spectrometer was used for the Raman measurement. Measurement range of the Raman spectrometer is 300~2400cm 1. The excitation wavelength of the laser beam that excites the substrate for analysis was 785 nm, and the detector was measured using a CCD V and the number of integrations was 1 second X 5 (total 5 seconds). As a measurement method, a method using a commercially available probe was adopted. A pyridine aqueous solution was used as a sample. Pyridine aqueous solution is an enhanced confirmation component widely used in surface enhanced Raman spectroscopy (SERS) research. Ultrapure water was used for the preparation of the pyridine aqueous solution. A commercially available slab optical waveguide is used for thin film measurement. In addition, in the Raman measurement using an optical waveguide, in addition to the above conditions, the measurement is performed at an incident angle of 40 ° and the number of integrations of 1 second X 10 (10 seconds in total). The optical waveguide was deposited on the opposite end face of the signal detector so that the laser beam was reflected, and then the upper face was deposited by the above gold deposition method.
[0035] 図 15に、一例として、 1Mのピリジン水溶液に対する通常 (増強回数 0回）ラマンス ベクトル (A)、 10— 4Mピリジン水溶液の通常ラマンスペクトル（B— 1)および多重増強 ラマンスペクトル (増強回数 3回）による増強スペクトル (B— 2)を示す。これらは実際 の測定前に円筒状ガラス内に純水を充填してラマンスペクトルを得て、試料のラマン スペクトル力も差し引くことでバックグランド補正を行っている。図 15に示すように、 1 Mのピリジン水溶液の通常ラマンスペクトル (A)ではピークが観察される。し力し、 10_ 4Mピリジン水溶液の通常ラマンスペクトル (B— 1)では濃度が薄いため、何もピーク は認められない。一方、多重増強回数 3回による 10— 4Mのピリジン水溶液 (B—1)で は、高濃度の水溶液のラマンスペクトル (A)と同様にラマンシフトのピークが認められ 、ラマン散乱の増強が確認された。 [0035] Figure 15, as an example, normal (enhanced zero impressions) for aqueous pyridine solution 1M Ramansu vector (A), 10- 4 normal Raman spectrum M aqueous pyridine (B- 1) and multiple enhancement Raman spectrum (enhancement The enhanced spectrum (B-2) by 3 times is shown. Prior to actual measurement, pure water is filled into a cylindrical glass to obtain a Raman spectrum, and the background spectrum is corrected by subtracting the Raman spectrum force of the sample. As shown in FIG. 15, a peak is observed in the normal Raman spectrum (A) of a 1 M aqueous pyridine solution. 10_ In the normal Raman spectrum (B-1) of 4 M pyridine aqueous solution, since the concentration is low, no peak is observed. On the other hand, in accordance with 10- 4 M pyridine aqueous solution (B-1) is multiplexed enhanced number 3 times, as well as the peak of the Raman shift observed Raman spectrum of a high concentration of the aqueous solution (A), enhancement of Raman scattering is confirmed It was done.
[0036] ピリジン水溶液のラマンスペクトルにおけるピーク強度と濃度の関係については、各 増強または多重増強回数のラマン測定において、ピリジンの C H面内変角振動に 帰属するラマンシフト 1013cm 1ピーク強度を X軸に、その時の濃度を Y軸にプロットし 検量線を作成することにより求めた。（図 18参照)。ラマンシフト 1013cm 1のラマンピ ーク強度は下記の式で算出した。 [0036] Regarding the relationship between the peak intensity and the concentration in the Raman spectrum of an aqueous pyridine solution, the Raman shift 1013cm 1 peak intensity attributed to the CH in-plane inflection vibration of pyridine on the X-axis in the Raman measurement of each enhancement or multiple enhancement frequency. The concentration at that time was obtained by plotting on the Y axis and creating a calibration curve. (See Figure 18). The Raman peak intensity at a Raman shift of 1013 cm 1 was calculated by the following formula.
[0037] ピーク強度 =〔ラマンシフト 1013cm 1散乱強度 (ピリジンの C—H面内変角振動）〕 〔ラマンシフト 1013cm 1ピーク両端の散乱強度の平均値〕 その結果、ピーク強度の増加に伴う、濃度の増加が認められ、定量性の存在が確 認された。そこで、それぞれの増強および多重増強回数において検出限界濃度と検 出感度増強度を算出した。検出限界濃度は SZN (signal to noise ratio) = 3におけ るピリジン水溶液の濃度として計算を行った。ノイズ値は、ラマンスペクトルにおいて ピークが何も認められな！ヽ 2300— 2350cm 1における二乗平均平方根（RMS)を用 、 た。得られた検出限界シグナル値は図 18の検量線と照らし合わせて検出限界濃度 を計算した。検出限界増強度は増強回数 0回における検出限界濃度を 1と設定し算 出した。その結果を図 16および図 17に示す。その結果、通常のラマン測定 (増強回 数 0回）では検出限界が 220ppmであるのに対し、 SERS (増強回数 1回）では 129pp bとなり、検出感度増強度は 1,700倍となった（図 16参照)。一方、 MERS (多重増強 回数 5回）では 0.96ppbとなり、通常のラマン測定に対して検出感度増強度は 209,900 倍となった（図 17参照)。つまり、 MERSを用いることにより通常のラマン分光計によ る検出感度に対して 10,000倍を超えるラマン散乱の増強が確認された。 [0037] Peak intensity = [Raman shift 1013 cm 1 scattering intensity (C—H in-plane bending vibration of pyridine)] [Raman shift 1013 cm 1 average of scattering intensity at both ends of peak] As a result, as the peak intensity increases, An increase in concentration was observed, confirming the existence of quantitativeness. Therefore, the detection limit concentration and the detection sensitivity enhancement intensity were calculated for each enhancement and multiple enhancement times. The detection limit concentration was calculated as the concentration of pyridine aqueous solution at SZN (signal to noise ratio) = 3. The noise value shows no peaks in the Raman spectrum!ヽ The root mean square (RMS) at 2300-2350 cm 1 was used. The obtained detection limit signal value was compared with the calibration curve in Fig. 18 to calculate the detection limit concentration. The detection limit enhancement was calculated by setting the detection limit concentration to 1 when the number of enhancements was zero. The results are shown in FIG. 16 and FIG. As a result, the detection limit for normal Raman measurement (0 enhancements) was 220 ppm, whereas for SERS (1 enhancement), the detection limit was 129 ppb, increasing the detection sensitivity by 1,700 (Fig. 16). reference). On the other hand, MERS (multiple enhancement times 5 times) was 0.96 ppb, and the detection sensitivity enhancement was 209,900 times that of normal Raman measurement (see Fig. 17). In other words, by using MERS, the enhancement of Raman scattering exceeding 10,000 times the detection sensitivity of a normal Raman spectrometer was confirmed.
[0038] それらから、多重増強回数を増やすにつれ検出感度増強度が増カロしていることが わかった。これは、単一のレーザ光源により、複数回の SERS測定が同時に可能であ ることに加えて、一定容量の溶液中に複数の基板を配置することによる発光現象ゃラ マン散乱光との共鳴による増強も考えられる。また、測定分子が基板に吸着する機会 が増加するため、検出感度増強度は基板の枚数に単純に比例する以上に増加する ことが示唆された。以上から、多重増強ラマン分光法の有効性が確認された。 [0038] From these results, it was found that the detection sensitivity enhancement increased as the number of multiple enhancements increased. In addition to being able to perform multiple SERS measurements at the same time with a single laser light source, the light emission phenomenon caused by placing multiple substrates in a fixed volume of solution is resonant with Raman scattered light. It is also possible to reinforce. Opportunities for measurement molecules to be adsorbed to the substrate Therefore, the increase in detection sensitivity was suggested to increase more than simply proportional to the number of substrates. From the above, the effectiveness of multiple enhanced Raman spectroscopy was confirmed.
[0039] また、蒸着基板表面を非晶性フッ素榭脂 (商品名サイトップ)を用いサブミクロンォ ーダ一でコーティングした場合でも、ラマン散乱増強は認められ、コーティングが可能 であることが示唆された。  [0039] Further, even when the surface of the vapor deposition substrate was coated with submicron order using amorphous fluorine resin (trade name Cytop), Raman scattering enhancement was observed, suggesting that coating is possible. .
[0040] 本手法を用いて、内分泌撹乱物質として疑われており、残留農薬としての制限があ るカーバメイト系殺虫剤 CarbaryKl-naphthyl methyl carbamate)の測定を行った結果[0040] Using this method, the results of measurement of carbate-type insecticide (CarbaryKl-naphthyl methyl carbamate), which is suspected as an endocrine disruptor and has limitations as a residual pesticide.
、ピリジン水溶液と同等の感度増強を確認した。 The same sensitivity enhancement as that of the pyridine aqueous solution was confirmed.
[0041] また、本発明を用いて重金属の測定も行った。測定する重金属としては、 6価クロム を有する-クロム酸カリウム (K Cr 0 )を用いた。超純水を用い、適切な濃度に調製し [0041] Heavy metals were also measured using the present invention. As the heavy metal to be measured, -potassium chromate (K Cr 0) having hexavalent chromium was used. Use ultrapure water and adjust to an appropriate concentration.
2 2 7  2 2 7
測定に用いた。測定用基板は、市販の金属イオンコーターを用い、金を基板に蒸着 して作成した。ラマン測定は、一例として、市販されているプロセスラマン分光計 PI-2 00 (プロセスインスツルメンッ社製）を用いた。測定範囲 300-2400cm 励起波長 785 應、検出器 CCD、積算回数 1秒 X 5 (合計 5秒）にて測定を行い、測定方法としては、 一例として、市販されているプローブ (インフォトニックス社製)を用いる方法を採用し た。  Used for measurement. The measurement substrate was prepared by depositing gold on the substrate using a commercially available metal ion coater. For example, a commercially available process Raman spectrometer PI-200 (manufactured by Process Instruments) was used for the Raman measurement. Measurement range 300-2400cm Excitation wavelength 785, detector CCD, number of integrations 1 second X 5 (total 5 seconds), as a measurement method, for example, a commercially available probe (manufactured by Inphotonics) ) Was used.
[0042] 通常のラマン測定 (多重増強回数 0回）には、試料 (約 300 ml)をガラスチューブに 充填し測定を行った。また、 SERS測定 (多重増強回数 1回）には、ガラスチューブに 蒸着カバーガラスを入れ測定を行った。更に、 MERS測定 (多重増強回数 3回）には 試料 (約 300 ml)を蒸着ガラスチューブに充填し測定を行った。  [0042] For normal Raman measurement (multiple enhancement times 0), a sample (about 300 ml) was filled in a glass tube and measured. For SERS measurement (multiple enhancement times 1), a vapor deposition cover glass was placed in the glass tube. Furthermore, for MERS measurement (multiple enhancements 3 times), a sample (about 300 ml) was filled in a vapor-deposited glass tube and measured.
[0043] 図 19に、 0.4 Mの-クロム酸カリウム水溶液の通常ラマンスペクトル (A)、 10— 4 M-ク ロム酸カリウム水溶液の多重増強回数 3回による増強スペクトル (B-1：実線）、超純水 の多重増強回数 3回による増強スペクトル (B-2 :点線)および 10— 3 M-クロム酸力リウ ム水溶液の通常のラマンスペクトル（B- 3 :破線）を示す。図 19の B-2からラマン散乱 の増強が認められた。よって、ラマン散乱増強の確認された 803 cm 1の散乱強度と 濃度との関係をプロットし、検量線を作成し、各増強回数における検出限界と増強度 を算出した。検出限界は SZN比（signal to noise ratio) = 3として計算を行った。ノィ ズ値は、増強スペクトルにおいてピークが何も認められない 2300— 2350cm— 1におけ る二乗平均平方根 (RMS)を用いた。増強度は増強回数 0回における検出限界を 1と 設定し、各増強回数の検出限界を算出した。結果を図 20に示す。これらから、重金 属の MERS測定が可能であることがわかった。 In [0043] Figure 19, of 0.4 M - normal Raman spectrum of chromic acid aqueous potassium (A), 10- 4 M- chromic acid aqueous solution of potassium multiple enhanced number 3 times by enhancing the spectrum of (B-1: solid line), super multi enhancement number 3 times by enhancing the spectrum of pure water (B-2: dotted line) and 10- 3 M- chromic acid force Liu anhydrous solution of normal Raman spectrum (B- 3: broken line) shows a. B-2 in Fig. 19 showed an increase in Raman scattering. Therefore, the relationship between the scattering intensity and concentration of 803 cm 1 in which Raman scattering enhancement was confirmed was plotted, a calibration curve was created, and the detection limit and enhancement intensity at each number of enhancements were calculated. The detection limit was calculated with SZN ratio (signal to noise ratio) = 3. The noise value is at 2300-2350cm- 1 where no peaks are observed in the enhanced spectrum. The root mean square (RMS) was used. For the enhancement, the detection limit at 0 enhancements was set to 1, and the detection limit for each enhancement was calculated. The results are shown in FIG. From these, it was found that MERS measurement of heavy metals is possible.
[0044] 更に、本発明を用いて菌の測定も実施した。供試菌株としては、大腸菌（Esherichia coli 0157 H:7)および黄色ブドウ球菌（Staphylococcus aureus)を用いた。各菌を 37 °Cで一晩振とう培養後、培養液 10 ml力も遠心集菌 (4°C、 8000 g、 5分)と滅菌水によ る洗浄を 5回繰り返し行った。その後、滅菌水 1 mlに混合させ、適切な濃度に希釈し 測定に用いた。測定用基板は、市販の金属イオンコーターを用い、銀を基板に蒸着 して作成した。ラマン測定は、一例として、市販されているプロセスラマン分光計 PI-2 00 (プロセスインスツルメンッ社製）を用いた。測定範囲 300-2400cm 励起波長 785 應、検出器 CCD、積算回数 1秒 X 5 (合計 5秒）にて測定を行い、測定方法としては、 一例として、市販されているプローブ (インフォトニックス製)を用いる方法を採用した 。測定には、試料 300 ml用いた。  [0044] Furthermore, bacteria were also measured using the present invention. As test strains, Escherichia coli 0157 H: 7 and Staphylococcus aureus were used. After each culture was shaken overnight at 37 ° C, 10 ml of culture broth was repeatedly centrifuged (4 ° C, 8000 g, 5 minutes) and washed with sterile water 5 times. Then, it was mixed with 1 ml of sterilized water, diluted to an appropriate concentration, and used for measurement. The measurement substrate was prepared by depositing silver on the substrate using a commercially available metal ion coater. For example, a commercially available process Raman spectrometer PI-200 (manufactured by Process Instruments) was used for the Raman measurement. Measurement range 300-2400cm Excitation wavelength 785, detector CCD, number of integrations 1 second X 5 (total 5 seconds), and as a measurement method, as an example, a commercially available probe (manufactured by Inphotonics) The method using was adopted. A 300 ml sample was used for the measurement.
[0045] 図 21に MERSを用いた大腸菌（A)および黄色ブドウ球菌（B)のラマンスペクトルを 示す。図中の（A)において 603および 743cm— 1にピークが認められたのに対し、図中 の（B)において 567および 919cm— 1にピークが認められたことから、食中毒菌の MER S測定が可能であり、また大腸菌および黄色ブドウ球菌の判別が可能であることがわ かった。 FIG. 21 shows Raman spectra of Escherichia coli (A) and Staphylococcus aureus (B) using MERS. In (A) in the figure, peaks were observed at 603 and 743 cm- 1 , whereas in (B) in the figure, peaks were observed at 567 and 919 cm- 1 , so MERS measurement of food poisoning bacteria was performed. It was found that E. coli and Staphylococcus aureus could be distinguished.
[0046] この他、光導波路を用いた測定においては、ポリ塩ィ匕ビユリデンが主成分である食 品包装用ラップフィルムの測定を行った。その結果、フィルム表面の微量添加物の明 瞭なスペクトルを得ることができた。以上から、水溶液以外の固体表面微量物質の測 定も可能であることが示唆された。  [0046] In addition, in the measurement using the optical waveguide, a food packaging wrap film containing polysalt vinylidene as a main component was measured. As a result, a clear spectrum of trace additives on the film surface could be obtained. From the above, it was suggested that it is possible to measure solid surface trace substances other than aqueous solutions.
[0047] その他、 MERSを用いて、アミノ酸、タンパク質、核酸、毒素、菌類、ポリアミン、木 粉、紙、ポリイミド、黒鉛多環芳香族、ガス、プラスチック、導電性ポリマーなどが測定 可能であると考えられる。  [0047] In addition, it is considered that amino acids, proteins, nucleic acids, toxins, fungi, polyamines, wood flour, paper, polyimide, graphite polycyclic aromatics, gas, plastics, conductive polymers, etc. can be measured using MERS. It is done.
産業上の利用可能性  Industrial applicability
[0048] 低濃度の物質を感度良く分析することが可能となるため、環境、食料或いは医療分 野における様々な物質分析に利用することができる。具体的には、河川、農地、飲料 水、工業排水中の残留農薬'環境ホルモンなどの有害成分の迅速な検出や、生体 内のたんぱく質 ·ペプチド'菌の検出、更には大気中の有害成分の分析などに利用 できる。 [0048] Since it is possible to analyze a low-concentration substance with high sensitivity, it can be used for various substance analyzes in the environment, food, or medical fields. Specifically, rivers, farmland, beverages It can be used for the rapid detection of harmful components such as residual agricultural chemicals 'environmental hormones' in water and industrial wastewater, the detection of protein / peptide bacteria in vivo, and the analysis of harmful components in the atmosphere.

Claims

請求の範囲 The scope of the claims
[I] ラマン分光分析に用いる分析用基板であって、所定の透明基板と、この透明基板 の表面に不均一に付着した金属粒子とからなることを特徴とする分析用基板。  [I] An analytical substrate used for Raman spectroscopic analysis, which comprises a predetermined transparent substrate and metal particles that are unevenly adhered to the surface of the transparent substrate.
[2] 前記金属粒子は、前記透明基板上の所定の領域ではナノオーダーの間隔で密集 し、それ以外の領域ではマイクロオーダーの間隔で分散して 、ることを特徴とする請 求項 1に記載の分析用基板。  [2] Claim 1 is characterized in that the metal particles are densely packed at nano-order intervals in a predetermined region on the transparent substrate, and are dispersed at micro-order intervals in the other regions. The analytical substrate described.
[3] 前記金属粒子は金、銀、銅、白金、ノラジウム、アルミニウム、チタン又はコバルトか らなることを特徴とする請求項 1又は 2に記載の分析用基板。 [3] The analytical substrate according to claim 1 or 2, wherein the metal particles are made of gold, silver, copper, platinum, noradium, aluminum, titanium, or cobalt.
[4] 前記透明基板は、板状であることを特徴とする請求項 1〜3の何れか一項に記載の 分析用基板。 [4] The analysis substrate according to any one of [1] to [3], wherein the transparent substrate has a plate shape.
[5] 前記透明基板は、円筒状であることを特徴とする請求項 1〜3の何れか一項に記載 の分析用基板。  [5] The analysis substrate according to any one of [1] to [3], wherein the transparent substrate is cylindrical.
[6] 前記透明基板は、直方体状であることを特徴とする請求項 1〜3の何れか一項に記 載の分析用基板。  [6] The analysis substrate according to any one of [1] to [3], wherein the transparent substrate has a rectangular parallelepiped shape.
[7] 少なくとも 2枚の上記請求項 4に記載の板状の分析用基板を相互に平行に配列し たことを特徴とする分析用基板組合体。  [7] An analytical substrate assembly comprising at least two plate-shaped analytical substrates according to claim 4 arranged in parallel to each other.
[8] 上記請求項 5に記載の円筒状の分析用基板と、この円筒状の分析用基板の内部 に挿入される少なくとも 1枚の上記請求項 4に記載の板状の分析用基板とからなるこ とを特徴とする分析用基板組合体。 [8] From the cylindrical analysis substrate according to claim 5, and at least one plate-shaped analysis substrate according to claim 4, which is inserted into the cylindrical analysis substrate. An analytical substrate assembly characterized by that.
[9] 直径の異なる少なくとも 2つの上記請求項 5に記載の円筒状の分析用基板を備え、 直径の大きな分析用基板の内部に直径の小さな分析用基板を配置したことを特徴と する分析用基板組合体。 [9] An analytical substrate comprising at least two cylindrical analytical substrates according to claim 5 having different diameters, wherein the analytical substrate having a small diameter is disposed inside the analytical substrate having a large diameter. Board assembly.
[10] 上記請求項 6に記載の直方体状の分析用基板と、この直方体状の分析用基板の 内部に挿入される少なくとも 1枚の上記請求項 4に記載の板状の分析用基板とからな ることを特徴とする分析用基板組合体。 [10] From the rectangular parallelepiped analytical substrate according to claim 6 and at least one plate-shaped analytical substrate according to claim 4 inserted into the rectangular parallelepiped analytical substrate. A board assembly for analysis characterized by
[II] 前記複数の分析用基板には、相互に異なる金属粒子が付着されていることを特徴 とする請求項 6〜10の何れか一項に記載の分析用基板組合体。  [II] The analysis substrate combination according to any one of claims 6 to 10, wherein different metal particles are attached to the plurality of analysis substrates.
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